Effects of edaravone dexborneol on functional outcome and inflammatory response in patients with acute ischemic stroke

Background Edaravone dexborneol has been reported as an effective neuroprotective agent in the treatment of acute ischemic stroke (AIS). This study aimed at investigating the impact of edaravone dexborneol on functional outcomes and systematic inflammatory response in AIS patient. Methods All participants were recruited from the AISRNA study (registered 21/11/2019, NCT04175691 [ClinicalTrials.gov]) between January 2022 and December 2022. The AIS patients were divided into two groups based on whether they received the treatment of edaravone dexborneol (37.5 mg/12 hours, IV) within 48 h after stroke onset. Inflammatory response was determined by detecting levels of cytokines (interleukin-2 [IL-2], IL-4, IL-5, IL-8, IL-6, IL-10, IL-12p70, IL-17, tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], IFN-α, and IL-1β) within 14 days after stroke onset. Results Eighty-five AIS patients were included from the AISRNA study. Patients treated with edaravone dexborneol showed a significantly higher proportion of modified Rankin Scale score < 2 compared to those who did not receive this treatment (70.7% versus 47.8%; P = 0.031). Furthermore, individuals receiving edaravone dexborneol injection exhibited lower expression levels of interleukin (IL)-1β, IL-6, and IL-17, along with higher levels of IL-4 and IL-10 expression during the acute phase of ischemic stroke (P < 0.05). These trends were not observed for IL-2, IL-5, IL-8, IL-12p70, tumor necrosis factor-α, interferon-γ [IFN-γ], and IFN-α (P > 0.05). Conclusions Treatment with edaravone dexborneol resulted in a favorable functional outcome at 90 days post-stroke onset when compared to patients without this intervention; it also suppressed proinflammatory factors expression while increasing anti-inflammatory factors levels. Trial registration ClinicalTrials.gov NCT04175691. Registered November 21, 2019, https://www.clinicaltrials.gov/ct2/show/NCT04175691.


Background
Stroke is a major cause of acquired adult disability and mortality worldwide [1].Intravenous thrombolysis and mechanical thrombectomy are currently the two most efficacious therapeutic approaches for acute ischemic stroke (AIS).Despite significant advancements in reperfusion treatment for ischemic stroke, the rate of clinically ineffective reperfusion remains around 50% in AIS patients [2].Moreover, numerous clinical trials investigating neuroprotective agents have failed to demonstrate any clinical benefits [3].The SAINT I and II trials suggested that neuroprotective agent NXY-059 was ineffective for AIS treatment within 6 h after stroke onset [4].Nerinetide was demonstrated to fail to improve functional outcomes after endovascular therapy [5].The ALIAS trials also showed that 25% albumin (2 g/kg, IV) did not improve 90-day clinical outcomes and increased the incidence of pulmonary edema and intracerebral hemorrhage [6].Another neuroprotective agent (magnesium sulfate) within 2 h after stroke onset also failed to improve functional outcomes at 90 days [7].Therefore, there is an urgent need to identify an effective neuroprotective agent that can reduce disability and mortality rates in AIS management.
In this study, we aimed to investigate the expression profile of systemic inflammatory factors and clinical benefits following edaravone dexborneol treatment in the acute phase of AIS patients.

Study population
The informed consent was obtained from all individuals.The study protocol adhered to the Declaration of Helsinki and received approval from the Ethics Committee of Nanjing First Hospital, Nanjing Medical University.A total of 85 patients with AIS were prospectively enrolled in an observational study of the AISRNA study (www.clinicaltrials.gov,NCT04175691).All participants were recruited from the Department of Neurology at Nanjing First Hospital, Nanjing Medical University.Inclusion criteria for enrollment included: [1] confirmation of anterior circulation cerebral infarction through non-contrast computer tomography (NCCT) or magnetic resonance imaging (MRI); [2] treatment initiation with edaravone dexborneol within 48 h after stroke onset, or absence of treatment with edaravone dexborneol; [3] age between 18 and 80 years old; and [4] National Institute of Health Stroke Scale (NIHSS) score ranging from 4 to 24.Exclusion criteria consisted of: [1] received the treatment with intravenous thrombolysis or mechanical thrombectomy after stroke onset; [2] presence of infectious diseases upon admission; [3] modified Rankin Scale score > 2 before stroke onset; [4] immunosuppressive therapy or antibiotic treatment within the past four weeks; [5] concurrent malignant tumors or severe organ failure including kidney, liver, and heart failure; [6] dysphagia; [7] lack of informed consent.

Procedures
All patients underwent standardized treatment according to AIS guidelines [14].The standardized group included antiplatelet aggregation or anticoagulant therapy, statin therapy, and control of risk factors regarding AIS.The treatment group received intravenous infusion of edaravone dexborneol at a dose of 37.5 mg administered by neurological nurses every 12 h for 14 days or hospital discharge and the standardized treatment.

Clinical characteristics
Demographics, medical history, stroke severity evaluation by NIHSS score assessment criteria [15] as well as stroke etiology based on Trial of Org 10,172 in acute stroke treatment (TOAST) criteria [16] were prospectively collected.Functional outcomes were assessed by modified Rankin Scale [17].Table 1 provides detailed baseline characteristics of patients with AIS.

Statistical analysis
Baseline characteristics between the groups were compared by using the SPSS 20.0 system.Continuous variables including inflammatory factors are expressed as the mean ± standard deviation (SD) or median (interquartile range [IQR]), and comparisons were made using t-test or one-way ANOVA if applicable; otherwise, Mann-Whitney U test was used.Categorical variables are presented as frequency (percentage) and compared using chisquare test.A P value less than 0.05 was considered statistically significant.

Baseline characteristics
Among the 978 AIS patients screened from the AIS-RNA study between January 2022 and December 2022, a total of 893 patients were excluded.The primary exclusions included: intravenous thrombolysis (n = 216), endovascular therapy (n = 136), NIHSS scores < 4 or > 24 (n = 205), posterior circulation infarction (n = 68), and invalid blood samples (n = 126).Therefore, 85 patients (44 patients received the standardized treatment [standardized group] and 41 patients received edaravone dexborneol plus standardized treatment [treatment group]) were included in the final analysis (Fig. 1).No significant differences in baseline characteristics were observed between these two groups (Table 1).

Association of edaravone dexborneol with clinical outcomes
There was no significant difference in NIHSS scores on admission between the treatment group and standardized group (P = 0.826).However, the treatment group exhibited lower NIHSS scores compared to the standardized group on day 10-14 (P = 0.019).There were no significant differences in NIHSS scores on day 2-3 and day 5-7 between these two groups (P > 0.05, Table 2).Additionally, there were 29 (70.7%)patients in the treatment group and 21 (47.8%)patients in the standardized group with mRS score < 2 on day 90 (P = 0.031, Table 2; Fig. 2).However, we did not observe a significant difference between the treatment (n = 33, 80.5%) and standardized groups (n = 35, 79.5%) with mRS score ≤ 2 on day 90 (P = 0.914, Fig. 2).

Dynamic change of inflammatory factors after treatment
Given the role of inflammatory response in acute ischemic stroke [18,19], we explored the influence of edaravone dexborneol on inflammatory response during the acute phase of ischemic stroke.We collected four samples at different time points: admission, day 2-3, day 5-7, and day 10-14.The results showed no significant differences in 11 inflammatory factors between the two groups upon admission except for IL-4 (P > 0.05, Table 3).However, compared to the standard group, the treatment group had lower levels of IL-1β and IL-17 on day 2-3, and higher levels of IL-4 and IL-10 on day 2-3 (P < 0.05, Fig. 3A, C, G, and I).On day 5-7, high levels of IL-4 and IL-10 were observed in the treatment group (Fig. 3C and  G), but IL-6 and IL-17 exhibited an opposite effect in the treatment group (Fig. 3E and I).On day 10-14, we only found decreased levels of IL-6 in the treatment group (P < 0.001, Fig. 3E).

Discussion
The present study of AIS patients from the AISRNA study demonstrated an improvement role of edaravone dexborneol on functional outcome in acute ischemic stroke.Furthermore, edaravone dexborneol inhibited proinflammatory factors expression and increased antiinflammatory factors levels during the acute phase of ischemic stroke.Over the past ten years, a series of clinical studies focusing on neuroprotection have failed to show significant benefits for ischemic stroke [4,7,[20][21][22].However, a phase III clinical trial has shown that administering edaravone dexborneol within 48 h after stroke onset leads to improved functional outcomes at day 90 compared    to edaravone alone [9].Ischemic stroke results in damage through various pathways in brain ischemia.Previous drugs targeting neuroprotection only interfere with a single mechanism of brain damage.In contrast, edaravone dexborneol protected against ischemic injury by multifunctional cytoprotective pathways including inflammatory, excitotoxic, oxidative and apoptotic insults [8,9,23].This study also demonstrates that edaravone dexborneol improves functional outcomes at day 90 in patients with acute ischemic stroke.Poststroke inflammatory response is involved in brain injury [24].Our previous study has shown that inflammatory factors have predictive value for stroke progression in patients undergoing endovascular therapy [25].Edaravone has been reported to protect endothelial and neuronal cells during brain ischemia through suppressing inflammation and neurotoxicity [26,27].Bornel shows potential as a neuroprotective agent by suppressing reactive oxygen species generation and inhibiting adverse inflammatory responses while reducing NO and NO synthase levels [28].A series of clinical and animal studies have shown that edaravone dexborneol alleviated ischemic brain damage by multiple molecular mechanisms including its effect on inflammatory response [8,12,[29][30][31][32].Additionally, a previous study showed that edaravone dexborneol reduced the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in the APP/PS1 mice [33].Importantly, edaravone dexborneol also suppressed the production of IL-1β, IL-6 and TNF-α and inhibited macrophages polarization to alleviate ischemic injury [34].Another study demonstrated that edaravone dexborneol facilitated M2 polarization of microglia through inhibiting TL4/NF-κB pathway [35].Furthermore, edaravone dexborneol exerted neuroprotective effect by inhibiting NF-κB/NLRP3/GSDMD signaling pathway and inflammatory factors (IL-1β and IL-18) in experimental ischemic stroke [32].These studies suggested that edaravone dexborneol may suppress inflammatory response through regulating macrophages polarization and NLRP3 inflammasome.Our study demonstrated that edaravone dexborneol effectively inhibited proinflammatory factors and increased anti-inflammatory factors during the acute phase of ischemic stroke.
The strengths of our study include the exclusion of interference from intravenous thrombolysis and endovascular therapy on inflammatory response, as well as an opposite effect of edaravone dexborneol on proinflammatory and anti-inflammatory factors during acute phase of ischemic stroke.There are several limitations to acknowledge.Firstly, this study was an observational and single center.Secondly, whole blood samples were collected upon admission, on day 2-3, 5-7, and 10-14.Thus, blood sampling schedule was irregular.Thirdly, we only observed dynamic changes in inflammatory factors during the acute phase without exploring further potential mechanisms underlying the suppression of inflammatory response by edaravone dexborneol.Lastly, some patients received treatment approximately 48 h after stroke onset which may have influenced the levels of inflammatory factors on day 2-3 or day 5-7.

Conclusions
This study demonstrated that the 90-day good functional outcome favored patients treated with edaravone dexborneol who had lower levels of proinflammatory factors and higher levels of anti-inflammatory factors compared to patients without this treatment.

Fig. 1
Fig. 1 Flowchart of the enrolled patients NIHSS, National Institute of Health Stroke Scale; mRS, modified Rankin Scale Institute of Health Stroke Scale; IQR, interquartile range; mRS, modified Rankin scale D2-3 indicates day 2-3 after stroke onset; D5-7, day 5-7; D10-14, day 10-14; D90, day 90 Treatment group received edaravone dexborneol intravenous infusion of 37.5 mg/dose (once every 12 h) on the basis of the standardized group Standardized group received antiplatelet aggregation or anticoagulant therapy, statin therapy, and control of risk factors regarding ischemic stroke

Fig. 2 Fig. 3
Fig. 2 Distribution of the 90-day modified Rank Scale scores

Table 1
Baseline characteristics of the enrolled patients DM, diabetes mellitus; CAD, coronary artery disease; AF, atrial fibrillation; IS, ischemic stroke; TIA, transient ischemic attack; IQR, interquartile range; NIHSS, National Institute of Health Stroke Scale; LAA, large artery atherosclerosis; SAO, small artery occlusion; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; mRS, modified Rankin scale Treatment group received edaravone dexborneol intravenous infusion of 37.5 mg/dose (once every 12 h) on the basis of the standardized group Standardized group received antiplatelet aggregation or anticoagulant therapy, statin therapy, and control of risk factors regarding ischemic stroke Continuous variables are expressed as median (interquartile range) or mean ± standard deviation (SD).Categorical variables are expressed as frequency and percentage a According to the modified TOAST classification

Table 2
Association of edaravone dexborneol on clinical outcomes in patients with ischemic stroke

Table 3
Baseline levels of inflammatory factors in patients with ischemic stroke